Genetically Modified Plants Displaying Reduced Accumulation of Cadmium

ABSTRACT

The present invention relates to methods and means such as polynucleotides, recombinant expression cassettes and recombinant vectors, for reducing the accumulation of cadmium in a plant, and a transgenic plant expressing a variant of a plant P 1B -type ATPase of the Zn 2+ /Co 2+ /Cd 2+ /Pb 2+  subgroup, said transgenic plant displaying a reduced accumulation of cadmium in the aerial parts.

The present invention relates to means and methods for reducing theaccumulation of cadmium in a plant, in particular tobacco.

Large cultivated areas present rising cadmium concentrations. This isdue on the one hand to the natural high level of metal in certain soils,and on the other hand to the use of fertilizers (due to the presence ofcadmium in the phosphate rock used for their manufacture), the spread ofwastewater treatment station sludge, the field watering with urbanwastewaters and the laying of aerosols. In the near future, the cadmiumconcentration in cultivated soils should continue to strongly increasedue to the depletion of cadmiumless-phosphate mines used as fertilizers.As a consequence, the cadmium content found in plants, mainly thosecultivated for foodstuffs, could be more and more frequently over thestandards implemented by the different countries or recommended by theFood and Agriculture Organization of the United Nations. By way ofexample, this is the case for green salad, tomato, pepper, wheat and,particularly, tobacco. Regarding tobacco, this plant is known toaccumulate high level of cadmium in leaves; the cadmium concentration intobacco is reported in the literature as being 0.5-5 ppm.

Cadmium is a metallic element belonging to group IIB of the periodictable of elements. Due to its chemical toxicity, inhalation or ingestionof high level of cadmium can represent severe health risks for bothhuman and animals (including damage to the respiratory system, kidney orliver, and cancer).

Therefore, there is a need for plants that can grow in a soil containinghigh cadmium concentration, and that are able to have a low content ofthis harmful metal. While different strategies may be followed to reducecadmium concentration in plants, the production of transgenic plant mayalso contribute to obtain such plants.

A method for producing transgenic plants with enhanced resistance anddecreased uptake of cadmium has been proposed by Lee et al. (2003, PlantPhysiol. 133:589-96; and International Application No. WO 02/081707).The authors have obtained genetically modified Arabidopsis thalianaexpressing Escherichia coli heavy metal ZntA protein. The transgenicplants displayed improved resistance to Pb and Cd, and the shootsthereof had decreased Pb and Cd content compared to the wild type.

ZntA is a P_(1B)-type ATPase that confers to E. coli resistance to toxicconcentrations of divalent cations, such as Zn²⁺, Cd²⁺ and Pb²⁺, byactive efflux of these metal ions outside the cytoplasm (Rensing et al.,1997, PNAS, 94:14326-14331; Rensing et al., 1998, J Biol Chem.,273:32614-32617; Dutta et al., 2007, Biochemistry, 46:3692-3703).

P_(1B)-ATPases are transporters that use the energy liberated by theexergonic ATP hydrolysis reaction to translocate across membranes softmetal cations, such as Zn²⁺, Cd²⁺, Pb²⁺, Co²⁺, Cu⁺ and Ag⁺ (Inesi, 1985,Annu Rev Physiol., 47:573-601; Axelsen and Palmgren, 2001, PlantPhysiol., 126:696-706; and for review: Argüello et al., 2007, BioMetals,20:233-248). They are sometimes referred to as HMAs (for Heavy MetalATPases) or to as CPx-type ATPases. P_(1B)-ATPases are found inprokaryote and eukaryote organisms, including archaea, bacteria, yeasts,insects, plants and mammals. They all contain the DKTGT signature aminoacid sequence. P_(1B)-ATPases are divided into two main distinctsubgroups (clusters) based on the substrate cation selectivity andphylogenetic analyses (Rensing et al., 1999, J Bacteriol.,181:5891-5897). The Cu⁺ cluster P_(1B)-ATPases are involved in thetransport of Cu⁺ and Ag⁺, whereas the Zn²⁺ cluster P_(1B)-ATPasestransport Zn²⁺ and other heavy metals such as Co²⁺, Cd²⁺ and Pb²⁺(Axelsen and Palmgren, 2001, above-cited). The different subgroups ofP_(1B)-ATPases have distinct motifs of conserved amino acid in theirtransmembrane domains. By way of example, their sixth transmembranedomain contains a (C, S, T)P(C,H) motif depending on the cationselectively transported (Solioz and Vulpe, 1996, Trends Biochem Sci.,21:237-241; Rensing et al., 1998, above-cited; Williams et al., 2000,Biochim Biophys Acta., 1465:104-126; Dutta et al., 2007, above-cited).

In Arabidopsis thaliana, HMA2 and HMA4 proteins (AtHMA2 and AtHMA4respectively) are P_(1B)-type ATPases that cluster with theZn²⁺/Co²⁺/Cd²⁺/Pb²⁺ subgroup. AtHMA2 and AtHMA4 are localized in plantaat the plasma membrane and are involved in the transport of thesedivalent cations to the aerial parts of the plant (Hussain et al., 2004,Plant Cell., 16:1327-39, Verret et al., 2004, FEBS Lett., 576:306-312).Zinc is an essential micronutrient required by the plants, while cobalt,cadmium and lead exhibit toxic effects. The amino acid sequence ofAtHMA2 and AtHMA4 are available under accession numbers GI|12229675 andGI|12229637 respectively in the GenBank database; AtHMA4 amino acidsequence is reproduced herein as SEQ ID NO: 1. These proteins containthe conserved CPC motif (herein after also denoted C₁PC₂) in their sixthtransmembrane domain and the DKTGT motif in the soluble loop betweentheir sixth and seventh transmembrane domain (Argüello et al., 2007,above-cited). In AtHMA4, the first cysteine residue (C₁) is located atposition 357 of the amino acid sequence (Mills et al., 2003, Plant J.,35:164-76). Mills et al. (2003, previously cited) showed that expressionof AtHMA4 in Saccharomyces cerevisiae decreased the sensitivity of theyeast to Cd and conferred resistance to this metal ion. In a secondarticle published in 2005 (FEBS Letters, 579:783-791), Mills et al.showed that expression in S. cerevisiae of an AtHMA4 mutant in which thefirst cysteine (C₁) of the CPC motif (³⁵⁷C) was substituted by a glycine(AtHMA4-C357G) did not confer Cd and Zn resistance to yeast, i.e.,yeasts expressing AtHMA4-C357G were more sensitive to elevated levels ofCd and Zn than those expressing the wild type AtHMA4.

Transgenic A. thaliana overexpressing AtHMA4 have also been obtained(Verret et al., above-cited; and International Application No. WO2005/090583). Contrary to the transgenic A. thaliana expressing ZntA (E.coli P_(1B)-type ATPase that clusters the Zn²⁺ subgroup) obtained by Leeet al. (see above), the transgenic A. thaliana obtained by Verret et al.displayed an increase in the Zn and Cd shoot content compared to thewild type.

It emerges from the foregoing that expression or overexpression in atransgenic plant of a P_(1B)-type ATPase that clusters the Zn²⁺ subgroupcan lead to a different phenotype according to the prokaryote or plantorigin of the P_(1B)-type ATPase.

Within the framework of research that has lead to the present invention,the Inventors have found, unexpectedly, that S. cerevisiae expressing anAtHMA4 variant in which the first cysteine (C₁) residue of the conservedC₁PC₂ motif in the sixth transmembrane domain was substituted by aserine (AtHMA4-C357S), exhibited diminished Cd transport capabilitieswhile the Zn efflux was almost identical in comparison with the wildtype yeast. Then, the Inventors have shown that a transgenic A. thalianaexpressing the AtHMA4-C357S variant displayed a reduced uptake ofcadmium compared to the wild type, while, advantageously, homeostasis ofthe physiological Zn²⁺ micronutrient was unmodified.

Accordingly, in a first aspect, the present invention provides a methodfor reducing the accumulation of cadmium in the aerial parts of a plant,characterized in that said method comprises expressing in said plant avariant of a plant P_(1B)-type ATPase of the Zn²⁺/Co²⁺/Cd²⁺/Pb²⁺subgroup having the C₁PC₂ motif in the sixth transmembrane domain, andlocalised in planta at the plasma membrane, said variant having amutation consisting of the substitution of the C₁ residue by any aminoacid selected from the group consisting of serine, alanine, histidineand threonine, preferably serine.

The term “aerial parts” includes, but is not limited to, the shoots,leaves, stems, flowers, fruits and seeds, preferably the leaves andseeds.

The localization of said P_(1B)-type ATPase in planta at the plasmamembrane can be determined by methods well-known from one of ordinaryskill in the art. By way of example, one can cite the methods describedin Hussain et al., 2004 and/or Verret et al., 2004 (both above-cited).Briefly, total plasma membranes from plants are fractioned by aqueoustwo-phase partitioning and the fractions are characterized by proteingel blot analysis probed with P_(1B)-type ATPase specific antibodies. Toconfirm the plasma membrane localization of said P_(1B)-type ATPase, thecoding sequence thereof can be fused in frame with a marker enzyme, suchas the green fluorescent protein (GFP) and expressed under control ofthe constitutive promoter, such as the CaMV 35S promoter, in transgenicplants or in protoplasts (transient expression). The subcellularlocalization of the P_(1B)-type ATPase-marker enzyme in cells can thenbe visualized by confocal microscopy.

According to a preferred embodiment of the invention, said P_(1B)-typeATPase is from a higher plant, such as A. thaliana, Nicotiana, Oryza andPopulus, and more preferably from the same plant species than the plantin which said expression is desired.

In another preferred embodiment, said P_(1B)-type ATPase is selectedfrom the group consisting of HMA4 and HMA2 from Arabidopsis thaliana,HMA4 from Thlaspi caerulescens (available under accession numberGI|46361990 in the GenBank database), HMA4 from Arabidopsis hallerisubsp. gemmifera (available under accession number GI|63056225 in theGenBank database), HMA2 and HMA3 from Oryza sativa (available underaccession numbers GI|125598398 and GI|125557764 respectively in theGenBank database) and HMA from Vitis vinifera (available under accessionnumber GI|157357491 in the GenBank database).

According to another particular embodiment of the invention, saidP_(1B)-type ATPase has at least 50% preferably at least 54% and by orderof increasing preference, at least 56%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 97%, 98% and 99% sequence identity, or at least 70%,preferably 73% and by order of increasing preference, at least 79%, 80%,85%, 90%, 95%, 97%, 98% and 99% sequence similarity with the AtHMA4protein of SEQ ID NO: 1, provided that it contains the conserved C₁PC₂motif in the sixth transmembrane domain.

Unless otherwise specified, the protein sequence identity and similarityvalues provided herein are calculated using the BLASTP program underdefault parameters, on a comparison window including the whole sequenceof the proteins. Similarity calculations are performed using the scoringmatrix BLOSUM62.

Optionally, the substitutions defined above can be combined with one ormore other mutation(s) aiming at improving the activity of thesemutants.

According to another particular embodiment of the invention, saidvariant has the amino acid sequence SEQ ID NO: 2. This amino acidsequence corresponds to the wild type AtHMA4 protein (SEQ ID NO: 1) inwhich the first cysteine (C₁) residue of the conserved C₁PC₂ motif inthe sixth transmembrane domain of the protein is substituted by an aminoacid as described above (i.e., serine, alanine, histidine or threonine).

According to another embodiment of the invention, the method forreducing the accumulation of cadmium in a plant further comprises theinhibition in said plant of at least one, preferably all, endogenous(wild type) P_(1B)-type ATPase(s) of the Zn²⁺/Co²⁺/Cd²⁺/Pb²⁺ subgrouphaving the C₁PC₂ motif in the sixth transmembrane domain and localizedin planta at the plasma membrane.

The method according to this embodiment only concerns plants expressingsaid endogenous P_(1B)-type ATPase(s). Indeed, plants may express one orseveral endogenous P_(1B)-type ATPases as defined above. By way ofexample, A. thaliana expresses the P_(1B)-type ATPase HMA2 and HMA4.Therefore, when the method according to this embodiment is applied to A.thaliana, then the method comprises the inhibition of HMA2 and/or HMA4.

The inhibition of an endogenous P_(1B)-type ATPase can be obtainedeither by abolishing, blocking or decreasing its function, oradvantageously, by preventing or down-regulating the expression of itsgene.

By way of example, inhibition of said endogenous P_(1B)-type ATPases canbe obtained by mutagenesis of the corresponding genes or of theirpromoters, and selection of the mutants having partially or totally lostthe P_(1B)-type ATPase activity. For instance, a mutation within thecoding sequence can induce, depending on the nature of the mutation, theexpression of an inactive protein; in the same way, a mutation withinthe promoter sequence can induce a lack of expression of said endogenousP_(1B)-type ATPases, or decrease thereof.

Mutagenesis can be performed for instance by targeted deletion of theendogenous P_(1B)-type ATPase coding sequences or promoters, or of aportion thereof, or by targeted insertion of an exogenous sequencewithin said coding sequences or said promoter(s). It can also beperformed by random chemical or physical mutagenesis, followed byscreening of the mutants within the gene encoding said endogenousP_(1B)-type ATPases. Methods for high throughput mutagenesis andscreening are available in the art. One can cite, for example, themethod described in Hussain et al., 2004 (above-cited) in which a T-DNAwas inserted in the hma2 and hma4 alleles from A. thaliana and themutants were grown on a medium containing high concentration of Zn(these mutants having Zn deficiency).

Advantageously, the inhibition of said endogenous P_(1B)-type ATPases isobtained by silencing of the corresponding genes. Methods for genesilencing in plants are known in themselves in the art. For instance,one can mention by antisense inhibition or co-suppression. It is alsopossible to use ribozymes targeting the mRNA of said endogenousP_(1B)-type ATPases.

Preferred methods are those wherein post transcriptional gene silencingis induced by means of RNA interference (RNAi) targeting the genesencoding said endogenous P_(1B)-type ATPases to be silenced. Variousmethods and DNA constructs for delivery of RNAi are available in the art(for review: Watson et al., 2005, FEBS Letters, 579:5982-5987).

The instant invention also provides a variant of a P_(1B)-type ATPaseprotein, as defined above.

The instant invention also provides means for carrying out saidexpression.

Thus, the present invention provides polynucleotides encoding a variantof a P_(1B)-type ATPase as defined above. The polynucleotides of theinvention may be obtained by the well-known methods of recombinant DNAtechnology and/or of chemical DNA synthesis. These methods also allow tointroduce the desired substitution in a naturally occurring nucleotidesequence encoding a P_(1B)-type ATPase as defined above.

The present invention also provides recombinant expression cassettescomprising a polynucleotide encoding a variant of a P_(1B)-type ATPaseas defined above under control of a transcriptional promoter allowingthe regulation of said polynucleotide in a host cell.

According to a preferred embodiment, said transcriptional promoter isany promoter functional in a plant cell, i.e., capable of directingtranscription of a polynucleotide encoding a variant of a P_(1B)-typeATPase as defined above, in a plant cell. The choice of the moreappropriate promoter may depend in particular on the organ(s) ortissue(s) targeted for expression, and on the type of expression (i.e.,constitutive or inducible) that one wishes to obtain. A large choice ofpromoters suitable for expression of genes in plants, and in particularin tobacco, is available in the art. They can be obtained for instancefrom plants (e.g., A. thaliana and N. tabacum), plant viruses, orbacteria such as Agrobacterium. They include constitutive promoters,i.e. promoters which are active in most tissues and cells and under mostenvironmental conditions, tissue or cell specific promoters which areactive only or mainly in certain tissues (e.g., leaves) or certain celltypes, and inducible promoters that are activated by physical orchemical stimuli. They also include the promoter of said endogenousP_(1B)-type ATPase from the same plant species than the plant in whichsaid expression is desired. The sequence of the promoters can also berepeated two or more times (e.g., duplicated in tandem).

Non-limitative examples of constitutive promoters that are commonly usedare the well-known cauliflower mosaic virus (CaMV) 35S promoter and thenopaline synthase (Nos) promoter, the Cassava vein Mosaic Virus (CsVMV)promoter (Verdaguer et al., 1996, Plant Mol. Biol., 31:1129-39), therice actin (1 or 2) promoter followed by the rice actin intron (RAP-RAI)contained in the plasmid pAct1-F4 used for transgenic monocotyledonplants (McElroy et al., 1991, Mol. Gen. Genet., 231(1):150-160).

The expression cassettes generally also include a transcriptionalterminator, such as the 35S transcriptional terminator or Nos terminator(Depicker et al., 1982, J. Mol. Appl. Genet., 1:561-73). They may alsoinclude other regulatory sequences, such as transcription enhancersequences.

The recombinant expression cassettes of the invention can be inserted inan appropriate vector allowing genetic transformation of the genome of ahost cell.

Thus, the present invention also relates to recombinant vectorscontaining an expression cassette as defined above, the promoter of saidexpression cassette being preferably a promoter functional in a plantcell.

The present invention also provides host cells containing a recombinantexpression cassette or a recombinant vector as defined above.

The host cells of the present invention are prokaryotic cells oreukaryotic cells, preferably plant cells, and more preferably tobaccocells.

In another aspect, the present invention provides a method for producinga transgenic plant having a reduced cadmium accumulation. Said methodcomprises the following steps:

a) providing a plant cell containing a recombinant expression cassetteor a recombinant vector as defined above, and optionally in which theendogenous P_(1B)-type ATPase as defined above is inhibited, and

b) regenerating from the plant cell obtained in step a) a transgenicplant expressing a variant of a P_(1B)-type ATPase as defined above.

Owing to the use of a variant of a P_(1B)-type ATPase of plant origin,the method of the invention has the advantage that the transgenic plantsproduced contain a minimal content of xenogenetic elements or foreignsequences, which makes the expression of said variant more stable andmore efficient.

The invention also comprises plants genetically transformed by arecombinant expression cassette of the invention, and expressing avariant of a P_(1B)-type ATPase as defined above. Preferably, saidtransgenic plants are obtainable by a method of the invention. In saidtransgenic plants, a recombinant expression cassette of the invention iscomprised in one or several transgene(s) integrated (i.e., stablyintegrated) in the plant genome, so that it is passed onto successiveplant generations. Thus the transgenic plants of the invention includenot only the plants resulting from the initial transgenesis, but alsotheir descendants, as far as they contain a recombinant expressioncassette of the invention.

Advantageously, a transgenic plant of the invention expressing a variantof a P_(1B)-type ATPase as defined above displays a reduced uptake ofcadmium, particularly in the aerial parts, when compared with a plant ofthe same species devoid of said transgene.

Accordingly, the invention provides a transgenic plant or an isolatedorgan (such as seeds, leaves, flowers, roots, stems, ears, preferablyleaves) or tissue thereof comprising, stably integrated in its genome, arecombinant expression cassette comprising a polynucleotide encoding avariant of a P_(1B)-type ATPase as defined above.

The present invention applies to monocot- or dicotyledon plants ofagronomical interest, such as wheat, barley, corn, rape, rice, chard,spinach, lettuce, tomato, tobacco, preferably tobacco.

Tobacco leaves naturally accumulate and concentrate relatively highlevels of cadmium, which is volatilized during burning, and contributessignificantly to a smoker's exposure to cadmium. Advantageously, atobacco leaf from a transgenic plant of the invention has lower cadmiumcontent when compared to a wild type tobacco or a tobacco devoid of saidtransgene(s) defined above.

Accordingly, in another aspect, the present invention relates to the usea tobacco leaf from a transgenic plant of the invention for themanufacture of tobacco products including smoking products, such ascigarettes, cigars and shags, as well as smokeless products, such assnuffing, chewing, or sucking tobacco, and the like. Said products arealso encompassed by the present invention.

Foregoing and other objects and advantages of the invention will becomemore apparent from the following detailed description and accompanyingdrawings. It is to be understood however that this foregoing detaileddescription is exemplary only and is not restrictive of the invention.

DESCRIPTION OF THE DRAWINGS

FIG. 1: a) Growth of pYES-only, AtHMA4- and AtHMA4SPC-yeasttransformants in liquid media followed by measuring optical density (OD)at 600 nm. Yeast cells were grown at 30° C. for 48 h in the presence ofCd 80 μM. b-d) Liquid cultures were carried out in the same conditionsas in a). The cells were collected, washed one time with 10 mM EDTA thenwashed two times with demineralised water to remove the cadmium adsorbedon the yeast cell walls. The pellets were dried, weighted, mineralisedwith HNO₃ then the metal content was determined by ICP-AES; in b) and d)the yeast cultures were carried out in the presence of Cd 80 μM; in c)the yeast cultures were carried out in a controlled nutrient solution(1.5 μM Zn).

FIG. 2 shows the in vitro root growth measurement of A. thalianaWassilewskija (Ws) and transgenic A. thaliana overexpressing AtHMA4(CPC) or AtHMA4SPC(SPC). Plantlets were grown vertically on bactoagarnutrient solution in the absence (control) or in the presence of thefollowing metals, 20 μM Cd, 50 μM Zn or 200 μM Zn. The root lengthmeasurements were carried out 14 days after germination. The Figureindicates the mean values of 100-150 measures +/−SD.

EXAMPLE Characterization of Transgenic Yeast and Arabidopsis ThalianaExpressing a Variant Form of AtHMA4

1) Material and Method:

The HMA4 cDNA from Arabidopsis thaliana (accession number AF412-407 inthe GENBANK database, Marsh 10, 2005 version), cloned in the inducibleyeast vector pYES-GFP (Gravot et al., 2004, FEBS Lett., 561:22-28.), wasmodified by site-directed mutagenesis. The amino acid residue cysteineat position 357 in the sequence was substituted by a serine (clonepVF472) to obtain the amino acid sequence SEQ ID NO: 2 in which is Xaais the amino acid serine (named AtHMA4SPC).

This construction was transformed in the yeast mutant strainshypersensitive to Cd and Pb (ycf1) (Li et al., 1996, J Biol Chem.,271:6509-6517) and to Zn (zrc1) (MacDiarmid et al., 2003, J Biol Chem.,278:15065-15072) respectively (clones pVF4121 and pVF4136, respectively)for heterologous experiments.

The insert of pVF472 was adapted by PCR for cloning in a plant vectorpresenting the strong ectopic promoter CaMV35S (clones pAP4152 andpAP4154). The vector pAP4154 was introduced in Agrobacterium tumefaciensstrain AglI.

A. thaliana plants (ecotype Wassilewskija) were transfected by floraldip (Clough and Bent, 1998, Plant J., 16:735-743). The transformantswere selected on hygromycin. Several independent lines of homozygousplants presenting one insertion of the construction were phenotypicallycharacterized. Plants of wild type (Ws) and overexpressing the variantform of AtHMA4 (AtHMA4SPC) were grown on solid control or in a mediumcontaining various cations transported by AtHMA4. Seeds were germinatedin a controlled-environment (8 h photoperiod at 300 μmol m⁻² s⁻¹, 21° C.and 70% relative humidity). The root length was measured 14 days aftergermination. T3 plants (homozygous plants carrying a unique T-DNA) wereused for phenotypic characterization.

A. thaliana overexpressing AtHMA4 were obtained as described in Verretet al. 2004, FEBS Lett., 576:306-312.

2) Results:

2-1) Characterization of the Transfected Yeasts:

2-1-1) The wild type yeast Saccharomyces cerevisiae was transformed withthe empty vector (pYES), the cDNA encoding the wild type HMA4 from A.thaliana (AtHMA4, SEQ ID NO: 1), or the cDNA encoding a variant ofAtHMA4 where the cystein at position 357 has been substituted with aserine (AtHMA4SPC, SEQ ID NO: 2). Liquid cultures in the presence of Cd80 μM were carried out at 30° C. and the O.D. was followed at 600 nmduring 48 hours. In agreement with the results obtained by Verret et al.(2005, FEBS Lett., 579:1515-1522), a better growth of the yeasttransformed with the wild type AtHMA4 was observed in the presence of Cd80 μM. AtHMA4 induced an increased tolerance toward cadmium. Whentransformed with the variant form (AtHMA4SPC), the yeast did not presentthis increased tolerance and the growth was identical to the controlstrain. Results are shown in FIG. 1 a.

2-1-2) The metal contents in yeast were determined by ICP-AES asdescribed by Gravot et al. (2004, above-cited). Results are shown inFIG. 1 b-d.

As previously described by Verret et al., 2005 (above-cited), Cdconcentration in yeast expressing AtHMA4 was found greatly lower (60%)than the Cd concentration in the control strain (FIG. 1 b). AtHMA4 wasexpressed at the plasma membrane of yeasts and had a role in anATP-dependent Cd/Zn efflux.

The Cd concentration in yeast expressing AtHMA4SPC was halfway betweenboth other strains (27.5% lower than the control). A lower capability totransport and efflux Cd could explain this observation.

The Zn concentration was measured in two growth conditions: in nutrientsolution containing 1.5 μM Zn, and in the presence of Cd 80 μM. In bothcases, the Zn concentration was identical in AtHMA4- andAtHMA4SPC-transformed yeasts and lower than in control yeasts (FIGS. 1 cand 1 d).

These experiments show that the Zn transport function of AtHMA4 was notmodified by the C357S substitution, even in the presence of a toxicconcentration of Cd, whereas the Cd transport capability was diminished.

2-2) Characterization of the Transfected A. thaliana:

The plant tolerance toward the various metal transported by AtHMA4 orAtHMA4SPC was determined by the measure of the root length on bactoagarsolid medium. The root length was the same for both genotypes in controlcondition. Results are shown in FIG. 2.

In the presence of different concentrations of Zn, Co and Pb, the rootlength was greater for seedlings overexpressing AtHMA4SPC than for thewild type ones. These results were in agreement with the resultsobtained in yeast, and with the observations made by Verret et al.(above-cited) in plants overexpressing the native form of AtHMA4. Plantsoverexpressing the variant form of AtHMA4 (AtHMA4SPC) present, as theones overexpressing the native form of AtHMA4, an increased tolerancetoward these three metals. This is due to more important metal transportand translocation to their shoot capabilities.

In the presence of Cd 20 μM, the root length was the same for both lines(wild type and variant). The increased tolerance toward cadmium alsoobserved for the plants overexpressing AtHMA4 was lost. This result isin agreement with those obtained with yeasts.

To conclude, a variant of a P_(1B)-type ATPase of theZn²⁺/Co²⁺/Cd²⁺/Pb²⁺ subgroup of eukaryotic origin having the C₁PC₂ motifin the sixth transmembrane domain (e.g., AtHMA4), said variant having amutation consisting of the substitution of the C₁ residue by a serine,presents a modified metal transport specificity probably due to adecreased affinity toward cadmium. However, this property is unchangedtoward the other three transported metals Co, Pb and especially thephysiological Zn.

1. A method for reducing the accumulation of cadmium in the aerial partsof a plant, wherein said method comprises expressing in said plant avariant of a plant P_(1B)-type ATPase of the Zn²⁺/Co²⁺/Cd²⁺/Pb²⁺subgroup having the C₁PC₂ motif in the sixth transmembrane domain andlocalised in planta at the plasma membrane, said variant having amutation consisting of the substitution of the C₁ residue by any aminoacid selected from the group consisting of serine, alanine, histidineand threonine.
 2. The method according to claim 1, wherein saidP_(1B)-type ATPase is from a higher plant.
 3. The method according toclaim 2, wherein said P_(1B)-type ATPase is from the same plant speciesthan the plant for which said expression is desired.
 4. The methodaccording to claim 1, wherein said P_(1B)-type ATPase is selected fromthe group consisting of HMA4 and HMA2 from Arabidopsis thaliana, HMA4from Thlaspi caerulescens, HMA4 from Arabidopsis halleri subsp.gemmifera, HMA2 and HMA3 from Oryza sativa and HMA from Vitis vinifera.5. The method according to claim 4, wherein the variant of a P_(1B)-typeATPase has the amino acid sequence SEQ ID NO:
 2. 6. The method accordingto claim 1, wherein it further comprises the inhibition of at least oneendogenous P_(1B)-type ATPase of the Zn²⁺/Co²⁺/Cd²⁺/Pb²⁺ subgroup havingthe C₁PC₂ motif in the sixth transmembrane domain and localised inplanta at the plasma membrane.
 7. A variant of a plant P_(1B)-typeATPase of the Zn²⁺/Co²⁺/Cd²⁺/Pb²⁺ subgroup having the C₁PC₂ motif in thesixth transmembrane domain and localised in planta at the plasmamembrane, said variant having a mutation consisting of the substitutionof the C₁ residue by any amino acid selected from the group consistingof serine, alanine, histidine and threonine.
 8. A polynucleotideencoding a variant of a P_(1B)-type ATPase of claim
 7. 9. A recombinantexpression cassette, comprising a polynucleotide as defined in claim 8,under control of a transcriptional promoter.
 10. A recombinant vector,comprising a recombinant expression cassette as defined in claim 9, thepromoter of said expression cassette being a promoter functional in aplant cell.
 11. A host cell, comprising a recombinant expressioncassette of claim
 9. 12. A host cell according to claim 11, wherein itis a plant cell.
 13. A method for producing a transgenic plant having areduced cadmium accumulation, comprising the following steps: a)providing a plant cell of claim 12, in which the endogenous P_(1B)-typeATPase is inhibited, and b) regenerating from the plant cell obtained instep a) a transgenic plant expressing a variant of a P_(1B)-type ATPase.14. A transgenic plant produced by the method of claim
 13. 15. Atransgenic plant or an isolated organ thereof or tissue thereof,comprising, stably integrated in its genome, a recombinant expressioncassette as defined in claim
 9. 16. The transgenic plant according toclaim 14, wherein it is a transgenic tobacco plant.
 17. An isolatedorgan according to claim 15, wherein it is a tobacco leaf. 18.(canceled)
 19. A host cell comprising a recombinant vector of claim 10.